Bone Marrow Stromal Cell Transplantation MitigatesRadiation-Induced Gastrointestinal Syndrome in MiceSubhrajit Saha1, Payel Bhanja1, Rafi Kabarriti1, Laibin Liu1, Alan A. Alfieri1, Chandan Guha1,2*

1 Department of Radiation Oncology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, United States of America, 2 Department of

Pathology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, United States of America

Abstract

Background: Nuclear accidents and terrorism presents a serious threat for mass casualty. While bone-marrowtransplantation might mitigate hematopoietic syndrome, currently there are no approved medical countermeasures toalleviate radiation-induced gastrointestinal syndrome (RIGS), resulting from direct cytocidal effects on intestinal stem cells(ISC) and crypt stromal cells. We examined whether bone marrow-derived adherent stromal cell transplantation (BMSCT)could restitute irradiated intestinal stem cells niche and mitigate radiation-induced gastrointestinal syndrome.

Methodology/Principal Findings: Autologous bone marrow was cultured in mesenchymal basal medium and adherentcells were harvested for transplantation to C57Bl6 mice, 24 and 72 hours after lethal whole body irradiation (10.4 Gy) orabdominal irradiation (16–20 Gy) in a single fraction. Mesenchymal, endothelial and myeloid population were characterizedby flow cytometry. Intestinal crypt regeneration and absorptive function was assessed by histopathology and xyloseabsorption assay, respectively. In contrast to 100% mortality in irradiated controls, BMSCT mitigated RIGS and rescued micefrom radiation lethality after 18 Gy of abdominal irradiation or 10.4 Gy whole body irradiation with 100% survival (p,0.0007and p,0.0009 respectively) beyond 25 days. Transplantation of enriched myeloid and non-myeloid fractions failed toimprove survival. BMASCT induced ISC regeneration, restitution of the ISC niche and xylose absorption. Serum levels ofintestinal radioprotective factors, such as, R-Spondin1, KGF, PDGF and FGF2, and anti-inflammatory cytokines were elevated,while inflammatory cytokines were down regulated.

Conclusion/Significance: Mitigation of lethal intestinal injury, following high doses of irradiation, can be achieved byintravenous transplantation of marrow-derived stromal cells, including mesenchymal, endothelial and macrophage cellpopulation. BMASCT increases blood levels of intestinal growth factors and induces regeneration of the irradiated host ISCniche, thus providing a platform to discover potential radiation mitigators and protectors for acute radiation syndromes andchemo-radiation therapy of abdominal malignancies.

Citation: Saha S, Bhanja P, Kabarriti R, Liu L, Alfieri AA, et al. (2011) Bone Marrow Stromal Cell Transplantation Mitigates Radiation-Induced GastrointestinalSyndrome in Mice. PLoS ONE 6(9): e24072. doi:10.1371/journal.pone.0024072

Editor: Jan-Hendrik Niess, Ulm University, Germany

Received April 19, 2011; Accepted July 29, 2011; Published September 15, 2011

Copyright: � 2011 Saha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The work has been supported by 1 RC2 AI087612-01 and 1U19AI091175-01 on Centers for Medical Countermeasures against Radiation from theNational Institute of Allergy and Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation ofthe manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: cguhamd@pol.net

Introduction

Accidental or intended radiation exposure in a mass casualty

setting presents a serious and on-going threat. At radiation doses of

3 to 8 Gy, morbidity and lethality is primarily caused from

hematopoietic injury and victims can be rescued by bone marrow

transplantation (BMT). However, with exposure to larger doses,

victims suffer irreversible hematopoietic and gastrointestinal injury

and usually perish despite supportive care and BMT. While BMT

may have some benefit in mitigating hematopoietic syndrome,

currently there are no approved medical countermeasures to

alleviate radiation-induced gastrointestinal syndrome (RIGS).

RIGS results from a dose-dependent, direct cytocidal and

growth inhibitory effects of irradiation on the villous enterocytes,

crypt intestinal stem cells (ISC) [1,2,3], the stromal endothelial

cells [4] and the intestinal subepithelial myofibroblasts (ISEMF)

[5]. Subsequent loss of the mucosal barrier results in microbial

infection, septic shock and systemic inflammatory response

syndrome. The cells in the ISC niche, consisting of micovascular

endothelial cells, mesenchyme-derived ISEMF [5] and pericryptal

macrophages [6] provide critical growth factor/signals for ISC

regeneration and intestinal homeostasis [7]. Of these, ISEMF

continuously migrate upward from the crypt base to the villous tip

along with ISC and transit amplifying enterocytes, establishing

signaling crosstalk and regulating ISC self-renewal and differen-

tiation [5,8]. ISEMF interacts with pericryptal macrophages with

subsequent release of PGE2 that could reduce radiation-induced

apoptosis of enterocytes [9,10]. Pericryptal macrophages form

synapses with crypt stem cells and secretes growth factors to

stimulate ISC proliferation [6] upon activation of Toll-like

receptors sensing the entry of bacteria and other intestinal

pathogens.

Since RIGS results from a combination of radiation-induced

loss of crypt progenitors and stromal cells along with aberrant

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signaling in the ISC niche, we rationalized that the acute loss of

stromal cells in the ISC niche would require rapid compensation

of their functions. This could be best achieved with cell

replacement therapies that restore the ISC niche after irradiation

so that the stromal cells can secrete growth factors and provide

necessary signals for survival, repair and regeneration of the

irradiated intestine. Earlier reports demonstrated that donor bone

marrow-derived cells could contribute to multiple lineages in the

gastrointestinal tract and facilitate intestinal regeneration in

patients with graft-versus-host disease and ulcer [11] and in

animal models of colitis [12]. Because of ease in cell culture and its

ability to differentiate into multiple tissue lineages, transplantation

of bone marrow-derived mesenchymal stem cells (MSC) has been

a very attractive option for a wide range of clinical applications

[13], such as, severe treatment-resistant graft-versus-host diseases

of the gut [14]. Besides trans-differentiating into ISEMF and

stimulating ISC proliferation, MSC transplantation has also been

shown to reprogram host macrophages to induce an anti-

inflammatory response and thereby minimizing sepsis in a murine

model of colitis [15]. Intravenous injection of MSC resulted in

enhanced engraftment in irradiated organs, including, small

intestine with subsequent increase in the regeneration of the

intestinal epithelium and accelerated recovery of the villi post-

radiation in mice models [16]. Genetic modification of donor

MSCs with superoxide dismutase [17] or CXCR4 [18] transgene

augments the engraftment and mitigation of intestinal radiation

injury. However, till date, transplantation of whole bone marrow

or MSC has not been successful in ameliorating RIGS and

improve survival of mice that received .10 Gy of irradiation in a

single fraction [16,17,18]. We reasoned that the failure of cell-

based therapies in ameliorating RIGS after lethal doses of

irradiation is because of absence of important cellular components

of the ISC niche, including endothelial cells and macrophages, in

the donor MSC population. Since bone marrow could provide a

source of all the major cell types in the ISC niche, namely, ISEMF,

endothelial cells and macrophages, we amplified the stromal cell

population by culturing freshly isolated bone marrow cells in

mesenchymal basal medium and collected the adherent stromal

cells for transplantation into mice exposed to lethal doses of whole

body or abdominal irradiation. In this report, we demonstrate that

bone marrow-derived adherent stromal cell transplantation

(BMASCT), 24 hours following exposure to lethal AIR of 16–

20 Gy, stimulated ISC regeneration, restored the functional

integrity of the villi, dampened inflammatory response and

mitigated RIGS in C57Bl/6 mice.

Results

BMASCT mitigates RIGS and improves survival of miceafter lethal doses of irradiation

Mortality from acute radiation syndromes results from dose-

dependent radiation injury to various organs. While BMT is

effective in improving survival of mice exposed to doses up to 8–

9 Gy, it is relatively ineffective as the sole treatment with higher

doses of exposure. We have previously demonstrated that a whole

body exposure of $10.4 Gy induces RIGS and 100% mortality

within 10–15 days in C57Bl/6 mice [1]. In order to confirm that

RIGS is induced after exposure to a single fraction of Whole Body

Irradiation (WBI) of 10.4 Gy, we examined whether BMT can

improve the survival of C57Bl/6 mice. While 100% of the

untreated animals died within 10 days, animals receiving BMT

had only 20% survival (Fig. 1A), indicating that whole marrow

that contained primarily CD45+ve hematopoietic cells (Figure S1)

failed to rescue these animals from RIGS. We, then, examined

whether transplantation of bone marrow-derived stromal cells that

have been enriched for MSC, Endothelial Progenitor Cell (EPC)

and macrophages upon culture in mesenchymal basal medium

could mitigate radiation injury in these animals. Fig. 1A

demonstrates that BMASCT rescued 100% of the irradiated

animals (p,0.0009), indicating that stromal cell therapy may

provide factors to repair and regenerate the intestinal epithelium

damaged by irradiation.

To limit the exposure of the bone marrow to irradiation while

escalating the dose to intestine, we delivered Abdominal

Irradiation (AIR) (12–20 Gy) after shielding the thorax, head

and neck and extremities, as described previously [19,20]. AIR did

not significantly impact the peripheral blood count at day 5

(Figure S2) post-exposure, indicating that the bone marrow was

not severely damaged by AIR. Control animals that received

either, PBS, or culture medium died within 10 days after exposure

to AIR$16 Gy with characteristic signs and symptoms of RIGS,

including, diarrhea, black stools and weight loss. In contrast,

animals that received AIR+BMASCT had well-formed stools,

maintained body weight (24.160.7 g in AIR+BMASCT versus

16.2161.8 g in AIR cohort, p,0.001) and had 100% survival

beyond 25 days (18 Gy AIR, p,0.0007, Fig. 1B). At 20 Gy,

BMASCT rescued 40% of the animals with survival greater than

25 days, while irradiated animals without BMASCT died within 5

days (median survival time of AIR cohort, 360.5 d versus

AIR+BMASCT cohort, 1261.8 d; p,0.01, Fig. 1C). Transplan-

tation of CD45+ hematopoietic cell-enriched bone marrow

derived non-adherent cell (BMNAC) and whole bone marrow

cells failed to rescue AIR-treated mice (Fig. 1B–C,E & Figure S1),

indicating that stromal cells were responsible for the salvage of

RIGS.

Both myeloid and non-myeloid cell populations areneeded for RIGS mitigation

Flow cytometric analysis of donor cells demonstrated that Bone

marrow-derived adherent stromal cell (BMASC) population

included, primarily MSCs (CD105+CD452 41.2%61.8;

CD29+CD452 39.8%61.2), macrophages (CD11b+F480+19.2%61.2) and EPCs (CD133+CD34+CD4522.6%60.89) and

CD45+ hematopoietic cells (Fig. 1D). CD44 and Sca1 staining

further confirmed the presence of MSC population (Figure S3). To

evaluate the individual roles of CD11b+ macrophage-enriched

cells versus CD11b2 MSC-enriched stromal cell fraction (Figure

S4) in RIGS mitigation, BMASC population was fractionated by

cell sorting using anti-CD11b-magnetic beads, followed by

transplantation 24 hrs post-AIR. Transplantation of either the

macrophage-enriched (78.1%62.8 F480+ cells), MSC-deficient

(,1.5% CD105+ve cells) CD11b+ve BMASC or macrophage-

deficient (0.68%60.03 F480+ cells), MSC-enriched (68–71%

CD105+ve) CD11b2ve BMASC cell population mitigated only

30–40% of the animals irradiated with 18 Gy AIR (Fig. 2A–B,

Figure S4). Survival was salvaged to 100% when the CD11b+ and

CD11b2 populations were admixed and transplanted, indicating

that the combination of macrophages and bone marrow stromal

cells, including MSC and EPC fractions was necessary for RIGS

mitigation.

BMASCT induces structural and functional regenerationof intestine

Histomorphological evaluation after hematoxylin-eosin staining

demonstrated that the AIR+BMASCT-treated animals exhibited

an increase in the overall size of the crypts and maintained villous

length (Fig. 3A, Figure S5 & S10). The percentage of the BrdU+ve

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crypt epithelial cells synthesizing DNA was significantly enhanced

in this cohort of mice at 3.5 days post-irradiation (AIR+BMASCT,

42.8262.01 versus AIR, 23.4361.66; P,0.04; Fig. 3B and E).

The numbers of regenerative crypt microcolonies per unit

intestinal cross sectional area at 3.5 days post-irradiation serves

as a surrogate indicator of crypt regenerative response post-

irradiation [1,21,22]. The crypt microcolony count was increased

significantly in AIR+BMASCT cohort, compared with those that

received AIR alone (AIR+BMASCT, 12.561.2/mm versus AIR,

6.860.8, p,0.004, Fig. 3D), indicating intestinal regenerative

response following BMASCT. Consistent with the regenerative

response, immunohistological analysis demonstrated the presence

of nuclear b-catenin in the AIR+BMASCT-treated animals, while

cytosolic staining was predominant in the animals receiving AIR

(Fig. 3C), suggesting that BMASCT activates the Wnt b-catenin

pathway in crypt cells to stimulate proliferation post-irradiation.

We performed xylose absorption test and determined the

functional recovery of the intestinal villi in RIGS. Since xylose is

not metabolized in the body, serum xylose level is a good indicator

of the intestinal absorptive capacity in animals fed with a test dose

of xylose [1]. Compared to animals that received AIR alone,

xylose absorption was significantly improved in animals that re-

ceived BMASCT at 7 d post AIR (AIR+BMASCT, 7265.5 g/ml

vs. AIR, 3562.7 g/ml; p,0.004; Fig. 3F), indicating quick

functional restitution of the intestinal villi.

BMASCT promotes survival of irradiated Lgr5-positivecrypt base columnar cells

We examined the effect of AIR on the number of Lgr5-

EGFP+ve crypt base columnar cells, the putative ISC population

[3,23], in the jejunum of Lgr5-EGFP-IRES-creERT2 transgenic

mice by detecting EGFP expression using confocal microscopy.

While these cells are present at 1 d post-AIR, they are absent at

3.5 d post-AIR (Fig. 4A). Flow cytometric analysis confirmed the

gradual loss of Lgr5+ve crypt ISCs following irradiation exposure

(5.17%61.8 at 1 d vs. 0.89%60.15 at 3.5 d; p,0.001; Fig. 4B). In

contrast, BMASCT increased the number of Lgr5-EGFP+ve

CBCs at 3.5 d post-AIR (Fig. 4A). Flow cytometric analysis

confirmed that BMASCT increased the number of irradiated

Lgr5-GFP+ve crypt cells at 3.5 d post-AIR (9.27%61.75, vs.

0.89%60.15; (p,0.0003; Fig. 4B), possibly by providing signals

for survival and growth. This provides us with a potential window

Figure 1. A–C. BMASCT improves survival of C57Bl/6 mice following AIR. Kaplan-Meier survival analysis of mice (n = 25) receiving BMASCT,24 and 72 hrs after irradiation, showed 100% survival after (A) 10.4 Gy WBI (p,0.0006) and (B) 18 Gy AIR (p,0.0007); and 40% survival after (C) 20 GyAIR (p,0.01). Whole bone marrow, BMNAC and culture media failed to improve survival. D–E. Flow cytometric characterization. (D) BMASC and(E) BMNAC population using MSC-specific (CD105+CD452/CD29+CD452), macrophage-specific (CD11b+F480+) and endothelial-specific(CD133+CD34+CD452) markers.doi:10.1371/journal.pone.0024072.g001

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of radiation mitigation, whereby BMASCT rescued lethally

irradiated mice within 24 hrs of irradiation, but not after 72 hrs

(Figure S6).

BMASCT restores the ISEMF and pericryptal macrophagesin the irradiated ISC niche

ISEMF and pericryptal macrophages provide the epithelial–

mesenchymal cross-talk signals for growth, differentiation and cell

fate determination to ISCs [6,8,9]. Immunohistochemistry and

confocal microscopy demonstrated that 18 Gy AIR reduces the

number of a-SMA+, desmin2ve ISEMF (Fig. 5A) and F480+ve

pericryptal macrophages (Fig. 5B). BMASCT restored the a-

SMA+, desmin2 ISEMF (Fig. 5A) and increased the number of

pericryptal and subepithelial macrophages in the lamina propria

(AIR+BMASCT, 7266.4/hpf versus AIR, 1563.2/hpf; p,0.003;

Fig. 5B,C) of irradiated mice. Transplantation of the CD11b2ve

fraction of BMASC restored the ISEMF population (Fig. 5A),

whereas transplantation of the CD11b+ve fraction exhibited an

increase in the number of intestinal macrophages (p,0.006,

Fig. 5B,C), which further suggests that transplantation of both

CD11b+ and CD11b2 fractions restores the ISC niche for RIGS

mitigation.

BMASCT induces secretion of intestinal growth factorsand anti-inflammatory cytokines

We examined the engraftment and repopulation of the donor

cells in various organs by transplanting dipeptidyl peptidase IV

(DPPIV)-proficient BMASC in DPPIV-deficient C57Bl/6 host.

Although some DPPIV+ve donor cells were noted per intestinal

villi upon DPPIV immunohistochemistry (Figure S7 A–B), the

majority of the donor cells were lodged in the lungs (Figure S7 C–

D). We, therefore, hypothesized that the regeneration and repair

of the irradiated intestine is possibly mediated by paracrine growth

factors that were secreted by the donor BMASCs. Immunoblot

analysis of the serum of animals that received AIR+BMASCT

showed an increase in serum levels of R-spondin1, FGF2, PDGF-

B and KGF by 2–8 folds at 24 h post-BMASCT, compared to

animals that received AIR alone (Fig. 6A). Interestingly, animals

that received whole BMT did not show an increase in serum R-

spondin1 levels (Figure. S8). While KGF and R-spondin1 can

increase the proliferation of intestinal crypt cells [1,24], FGF2 and

PDGF-B could support the growth of endothelial cells [4] and

ISEMF [25], respectively in the ISC niche of AIR+BMASC-

treated animals.

RIGS is associated with a systemic inflammatory response

syndrome (SIRS) resulting from bacterial entry from the denuded

gut lumen and resultant endotoxemia [26]. We performed multi-

cytokine ELISA in the serum of animals that received AIR alone

and compared them with those that received AIR+BMASCT.

Compared to untreated controls, there was a significant increase in

serum pro-inflammatory cytokines, such as, IL12A (p,0.001),

IL17 (p,0.006) in animals that received AIR (Fig. 6C) or

AIR+BMT (Figure S9B). BMASCT reduced the secretion of these

inflammatory cytokines, while inducing the release of anti-

inflammatory cytokines, IL6 (p,0.004) and IL10 (p,0.002)

(Fig. 6B) that may dampen the SIRS in RIGS. AIR+BMASCT

also increased the levels of serum GCSF (p,0.006) and GMCSF

(p,0.007) (Fig. 6D) compared to AIR alone, which could induce

macrophage infiltration and activation in the irradiated intestine

(Fig. 5B).

Since BMASCT was postulated to modulate the ISC niche, we

also examined the expression of mRNA level of intestinal growth

factors and inflammatory cytokines from cells isolated from the

crypt region. Quantitative RT-PCR analysis of crypt cell mRNA

from AIR+BMASCT-treated animals showed several fold increase

in expression level of intestinal growth factors, such as, FGF10,

KGF, EGF, FGF2, and anti-inflammatory cytokine, IL-10 with

BMASCT at 24 hr post-AIR, compared to AIR alone (see Tables

S1, S2, S3). While R-spondin1 levels were elevated in the serum,

its expression was absent in the crypt region. In contrast to

BMASCT, whole BMT had lower expression of intestinal survival

and growth factors and chemokines, such as, EGF, FGF10, FGF,

Figure 2. Both myeloid and non-myeloid fractions of BMASC are needed for RIGS mitigation. A. Flow cytometry of macrophagepopulation in CD11b+ and CD11b2 BMASC. B. Kaplan-Meier survival analysis.doi:10.1371/journal.pone.0024072.g002

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IGF1, VEGFa, CSF1, CXCL1 and CXCL12 (Table S1). These

results suggested that bone marrow-derived stromal cells could

modulate the regenerative signals in intestinal microenvironment.

Depletion of host macrophages reduces survival ofAIR+BMASCT-treated mice

Pericryptal macrophages play an important role in forming

synapses with ISC and modulating ISC regeneration [6]. To

evaluate the involvement of host macrophages in RIGS mitigation,

we depleted them by administering clodronate-filled liposomes

(clodrosome) intraperitoneally from day 4 pre-AIR to a week post-

AIR. The depletion of macrophages (CD11b+F480+) was verified

using FACS analysis of splenocytes and immunohistochemical

staining of intestinal sections (Fig. 7B–C). Macrophage depletion

reduced the RIGS-mitigating effect of BMASCT with only 25% of

the animals surviving after 18 Gy AIR, compared to 100%

survival in mice that received AIR+BMASCT (Fig. 7A). This

indicated an essential role of host macrophages in the regenerative

process of irradiated intestines.

Prostaglandin E2 (PGE2) is an essential mediator forBMASCT-induced RIGS mitigation

Intestinal macrophages have been implicated in inducing the

expression of COX2 for PGE2 synthesis by ISEMF. PGE2 has

been known to be involved in selfrenewal and differentiation

process of hematopoetic stem cell (HSC). Furthermore, PGE2

increased the homing efficiency of HSCs with the selective

induction of short-term-HSC engraftment in murine models [27].

Moreover, it was shown that PGE2 also inhibits the radiation-

induced apoptosis of intestinal crypt cells by binding to the EP

receptor on ISC [9,10]. To further elaborate on the cross-talk of

pericryptal macrophages and ISEMF in the ISC niche that are

replenished after BMASCT and also involvement of PGE2 in

repair process, we inhibited PGE2 synthesis with COX2 inhibitor

NS398. COX2 inhibition reduced the BMASCT-mediated

survival of irradiated animals to 35% (p,0.008), which was

restored to 80% with dmPGE2 supplementation (Fig. 7D). Tunnel

staining demonstrated that COX2 inhibition significantly in-

creased the percent of apoptotic cell in crypt of animals that

received AIR+BMASCT (p,0.002) (Fig. 7 E–F), which was

reduced with dmPGE2 supplementation (Fig. 7 E–F).

Discussion

This is the first demonstration of RIGS mitigation by

BMASCT, 24 hours after exposure to high doses of either, single

fraction of whole body irradiation (10.4 Gy) or AIR (16–20 Gy).

BMASCT restores the ISC niche, including, the pericryptal

macrophages, endothelial cells and ISEMF. In contrast to BMT

that mitigates radiation-induced hematopoeitic syndrome by

donor cell repopulation, BMASCT mitigates RIGS via accelerated

regeneration of irradiated host ISC rather than its replacement

with donor derived cells. This would require the presence of Lgr5+

Figure 4. BMASCT promotes survival of Lgr5-positive crypt base columnar cells following AIR. A. Confocal microscopic imaging of EGFPexpression in the jejunum of Lgr5-EGFP-ires-CreERT2 transgenic mice. Lgr5-EGFP+ve crypt cells are present at 1 d post-AIR but are absent at 3.5 dpost-AIR, indicating the time course of radiation-induced ISC death. BMASCT inhibits the radiation-induced cell loss of Lgr5+ISCs. Confocalmicroscopic images (636) were magnified 2.36 (inset). Nucleus was stained with DAPI and pseudo colored with red. B. Flow cytometric analysis ofEGFP expression in crypt cells of Lgr5-EGFP-ires-CreERT2 transgenic mice post-AIR.doi:10.1371/journal.pone.0024072.g004

Figure 3. BMASCT mitigates RIGS by promoting structural and functional regeneration of the irradiated intestine. A. H&E staining, B.Brdu immunohistochemistry, C. b-catenin immunohistochemistry. b-catenin stained green and nucleus was stained with DAPI (pseudo colored withred). Confocal microscopic images (636) were magnified 2.36 (inset). Note the greater crypt depth (A), increase in crypt cell proliferation (B) and anincrease in nuclear translocation of b-catenin (stained yellow) in AIR+BMASCT cohort compared to other cohorts. D. Number of regenerative crypts,E. Crypt proliferation rate and F. Xylose absorption assay. A time course study showed significant recovery (p,0.0003) of xylose absorption at 7 dayspost-irradiation in AIR+BMASCT-treated animals compared to the AIR cohort.doi:10.1371/journal.pone.0024072.g003

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ISCs, which were noted in crypt for 24 hrs post-AIR, thus

affording a time window for effective radio-mitigation. Hence,

BMASCT was successful in rescuing animals up to 24 hrs post-

radiation but not at later time points.

Since the majority of the donor cells were lodged in the lungs,

radiation injury was perhaps mitigated by secreted growth factors.

Potential candidates include R-spondin1, KGF, FGF2, PDGF-B,

IL-6, IL-10, G-CSF and GM-CSF. Serum R-spondin1 levels

increased by 8–10-fold. Human R-spondin1, a 29 kd, 263 amino

acid protein that acts as a specific growth factor of intestinal crypt

cells [28], has been shown to be a mucosal protective agent in

radiation and chemotherapy-induced mucositis [29]. We have

demonstrated that R-spondin1 can be radioprotective for RIGS

[1]. R-spondin1 binds with high affinity to the Wnt co-receptor,

LRP6, and induce phosphorylation, stabilization and nuclear

translocation of cytosolic b-catenin, thereby activating TCF/b-

catenin-dependent transcriptional responses in intestinal crypt cells

[30]. The presence of nuclear b-catenin in the crypt cells of

AIR+BMASCT-treated animals could represent R-spondin1-

mediated Wnt activation in ISC of these animals. BMASCT also

modulated the mRNA expression of several intestinal growth

factors in the crypt cells of irradiated intestine. However, R-

spondin1 was not expressed in the cells of the crypt region.

BMT can rescue animals that develop primarily a hematopoi-

etic syndrome with exposure to radiation doses #8–9 Gy in single

fraction. With higher doses of irradiation, intestinal injury sets in

and animals cannot be rescued by BMT alone. Although, bone

marrow-derived, MSCs contribute to intestinal regeneration and

transplantation of these cells ameliorated intestinal injury in

murine models of radiation and chemotherapy-induced injury,

colitis, and autoimmune enteropathy [16,18,31,32], MSC trans-

plantation alone failed to improve survival of animals exposed to

higher irradiation doses (.9.6 Gy) in a single fraction [16,17,18].

Our study shows that whole bone marrow transplantation cannot

mitigate intestinal injury induced by irradiation ($10.4 Gy).

However, upon amplification of stromal cells in mesenchymal

basal medium culture, and transplantation of a combination of

CD11b+ macrophages and CD11b2 MSC and EPCs could

Figure 5. BMASCT restores the ISEMF and pericryptal macrophages of the ISC niche, 3.5 days post-AIR. A. ISEMF detection byimmunohistochemistry and confocal microscopy using anti-a-SMA (stained red, indicated with arrow) and anti-desmin (stained green) antibodies. a-SMA+ve and desmin2ve ISEMF were reduced in AIR-treated animals, which was restored by BMASCT. Nucleus was stained with DAPI (blue). B. F480Immunhistochemistry and confocal microscopic analysis and C. Quantification of Number of pericryptal macrophages. The number of F480+vemacrophages (green, indicated with arrow) increased at 3.5 d post-AIR in the AIR+BMASCT (p,0.003) and CD11b+ve BMASCT (p,0.006) group,compared to the AIR cohort, respectively. Nucleus was stained with DAPI (pseudo colored with red). Confocal microscopic images (636) weremagnified 2.36 (inset).doi:10.1371/journal.pone.0024072.g005

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effectively mitigate RIGS. Important differences were noted in the

animals that received BMASCT from BMT. In contrast to the

AIR+BMT cohort, the AIR+BMASCT cohorts had elevated

levels of serum R-spondin1 and expressed various intestinal

growth factors in the crypt cells, suggesting a role of stromal cells in

secreting growth factors and signals for inducing ISC proliferation

in these animals. These stromal cells secrete factors that support

the regeneration of the ISC and its niche. Increased serum levels of

Figure 6. Serum analysis of intestinal growth factors and cytokines. A. Immunoblot analysis. An increase in the serum levels of R-spondin1,FGF2, KGF and PDGF-B was noted in AIR+BMASCT cohort compared to AIR. B–D. Multi cytokine ELISA. B. Anti-inflammatory cytokines, IL6 (p,0.004)and IL10 (p,0.002) levels were significantly increased in the AIR+BMASCT, cohort compared to AIR alone. C. Pro-inflammatory cytokines, IL12A(p,0.001) and IL17 (p,0.006) levels were induced in AIR cohort, compared to AIR+BMASCT treated animals (IL12A, p,0.001; IL17, p,0.008). D.Myeloid cytokines, GM-CSF (p,0.007) and G-CSF (p,0.006) were increased in AIR+BMASCT group, compared to AIR.doi:10.1371/journal.pone.0024072.g006

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PDGF-B and FGF2, growth factors for ISEMF and EPC

proliferation [25], along with GMCSF and GCSF [33,34] for

macrophage activation support the involvement of BMASC in

restoring the ISC niche. Several growth factors that could mediate

intestinal regeneration, such as, FGF10, FGF, EGF, IGF1,

VEGFa, CSF1 and CXCL12 were induced in the crypt cells in

BMASCT-transplanted animals. ISEMF residing throughout the

lamina propria and pericryptal region plays a vital role in intestinal

structural regeneration [7,8,25]. Similarly, submucosal macro-

phages are activated by the bacterial ligands for Toll-like receptors

(TLR) upon bacterial entry through disrupted intestinal mucosa.

Thus activated macrophages act as ‘‘mobile cellular transceivers’’

that transmit regenerative signals to ISCs [6]. Crosstalk between

host macrophages and ISEMF was necessary for RIGS mitigation

by PGE2-mediated inhibition of radiation-induced apoptosis of

crypt cells, also noted in other studies [9,10]. Regenerative role of

PGE2 is very well established in hematopoetic system where it was

reportedly involved in engraftment as well as survival of

transplanted HSCs or cord blood cells [27,35]. Moreover in

embryonic and adult zebrafish model it was shown that PGE2 is

required for Wnt-mediated effects on HSC development and can

enhance Wnt activity in-vivo [27,36]. It was quiet evident in our

observation that PGE2 has a significant role in BMASCT-

mediated amelioration of RIGS. Based upon previous studies

[27,35], it is possible that PGE2 could increase the engraftment of

stromal cells. Furthermore, PGE2 from ISC niche may induce

Wnt signaling in ISCs, thereby participating in intestinal

regeneration [27,36].

In summary, these experiments point towards a new paradigm

for RIGS mitigation, whereby growth factors secreted after

BMASCT induce regeneration of the irradiated host crypt

progenitors and ISC niche, thereby, accelerating functional

recovery of the intestine in RIGS. By reducing the levels of pro-

inflammatory cytokines, while inducing anti-inflammatory cyto-

kines, BMASCT also dampens the SIRS in RIGS. Thus,

BMASCT provides a platform to discover potential biological

agents for mitigation of acute radiation syndromes and for mucosal

radioprotection during chemoradiation therapy of abdominal

malignancies.

Materials and Methods

AnimalsFive- to 6-weeks-old male C57Bl/6 (NCI-Fort Dietrich, MD),

dipeptidyl peptidase-deficient (DPPIV2ve) (gift from Dr. David

Shafritz, Einstein College, Bronx, NY) Lgr5-EGFP-IRES-creERT2

(Jackson Laboratories, Bar Harbor, Maine) mice were maintained

ad libitum and all studies were performed under the guidelines and

protocols of the Institutional Animal Care and Use Committee of

the Albert Einstein College of Medicine. The animal use protocol

for this study was reviewed and approved by the Institutional

Animal Care and Use Committee (IACUC) of Albert Einstein

College of Medicine (IACUC approval# 20080703).

IrradiationIrradiation was performed on anesthetized mice (intraperitoneal

ketamine and xylazine 7:1 mg/ml for 100 ml/mouse) using a

320 KvP, Phillips MGC-40 Orthovoltage irradiator at a 50 cm

SSD with a 2 mm copper filter at a dose rate of 72 cGy/min. We

administered WBI (10.4 Gy) or escalating doses of AIR (16–

20 Gy) after shielding the thorax, head and neck and extremities

and protecting a significant portion of the bone marrow, thus

inducing predominantly RIGS.

BMASC transplantationDonor bone marrow cells were harvested using sterile techniques

from the long bones from C57Bl/6 mice and cultured in MSC basal

medium (Cambrex-Lonza, Walkersville, MD) supplemented with

10% heat inactivated FBS, 1% Glutamine, and 1% Penicillin/

Streptomycin for 4 days, followed by collection of adherent

cells as BMASC. BMASC were then subjected to flow cytome-

tric characterization to determine the percentage of MSC

(CD105+CD452/CD29+CD452), EPC (CD34+CD133+CD452)

and macrophages (CD11b+ F480+). CD11b+ve and CD11b2ve

cells were fractionated using anti-CD11b-magnetic beads (MACS,

Miltenyi Biotec, Auburn, CA), following the manufacturer’s

protocol. Fractionated and whole BMASC (26106 cells/mice) were

injected intravenously via tail vein to C57Bl6 mice at 24 and

72 s hours after irradiation.

Characterization of RIGSAnimals were sacrificed at 1, 3.5 and 7 days after irradiation for

histopathological analysis to examine apoptosis by TUNEL

staining, regenerating crypt colonies and villi denudation (Hema-

toxylin and eosin staining) [1]. To visualize villous cell prolifer-

ation, each mouse was injected intraperitoneally with 120 mg/kg

BrdU (Sigma-Aldrich, USA) 2–4 hrs prior to sacrifice and mid-

jejunum was harvested for paraffin embedding and BrdU

immunohistochemistry (Text S1). The crypt proliferation rate

was calculated as the percentage of BrdU positive cells over the

total number of cells in each crypt. A total of 30 crypts were

examined per animal for all histological parameters. A regener-

ative crypt was confirmed by immunohistochemical detection of

BrdU incorporation into five or more epithelial cells within each

crypt, scored in a minimum of four cross-sections per mouse. The

number of regenerative crypts was counted for each dose of

irradiation and represented using the crypt microcolony assay

[1,21,22].

Characterization of ISCLgr5+ve ISCs were detected in 4% para-formaldehyde-fixed

sections from Lgr5-EGFP-ires-CreERT2 mouse jejunum by exam-

ining EGFP expression using confocal microscopy, according to

published protcols [3]. GFP expression was also measured by flow

cytometry of crypt cells, isolated from Lgr5-EGFP-ires-CreERT2

mouse intestines, according to method described earlier [23].

Figure 7. BMASCT promotes signaling cross-talk between macrophages and ISEMF in the ISC niche post-AIR. A. Kaplan-Meier survivalanalysis of animals treated with AIR+BMASCT following depletion of host macrophages by clodrosome. Clodrosome treatment reduced the animalsurvival after AIR+BMASCT to 25%, indicating host macrophages are needed for mitigation. B–C. Flow cytometric (B) and confocal microscopicevaluation (C) of macrophages. Note depletion of host macrophages post-AIR by clodrosome. D–F. Inhibition of COX2 reduced the BMASCTmediated mitigation of RIGS. D. Kaplan-Meier survival analysis. Administration of COX2 inhibitor, NS398, reduced survival of animals treated withAIR+BMASCT (p,0.008). Survival was improved to 80% with dmPGE2 supplement. E–F. TUNEL staining of crypts. AIR+BMASCT inhibited apoptosis inthe crypts at day 3.5, which was increased by NS398-mediated COX2 inhibition (p,0.002). Supplementation with dmPGE2 restored the anti-apoptoticeffect of BMASCT (p,0.005).doi:10.1371/journal.pone.0024072.g007

Stromal Cell Transplantation Mitigates RIGS

PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24072

Characterization of ISC nicheISEMF were stained in formalin-fixed, paraffin-embedded

tissue sections for alpha-smooth muscle actin (a-SMA) and desmin

using Cy3-conjugated mouse anti-a-SMA (1:500; Sigma, St. Louis,

MO) and rabbit anti-desmin (1:250; Abcam, Cambridge MA)

antibody, respectively, with overnight incubation at 4uC followed

by staining with goat anti-rabbit Alexafluor 488 (1:1000;

Molecular Probes, Carlsbad, CA). Pericryptal macrophages were

stained by Alexa Fluor488-conjugated, rat anti-mouse, F480

antibody (1:50; Caltag laboratories, Carlsbad, CA). Images were

captured using a Zeiss SP2 confocal microscope at 636 optical

zoom and the macrophages were counted by using the

VelocitySoft Version 5.0 (Improvision, Waltham, MA) in 10 fields

per mice in various cohorts (n = 3).

Intestinal absorptionFunctional regeneration of the irradiated intestines was

determined by measuring intestinal absorption by a xylose uptake

assay [1,37]. Briefly, 5% w/v D-xylose solution was administered

orally by feeding tube (100 mL/mice, n = 5/cohort), followed by

collection of blood 2 hours post-feeding. Plasma xylose levels were

measured by a modified micro-method [37].

Cytokines and growth factors in bloodIntestinal growth factors, R-spondin1, keratinocyte growth

factor (KGF), basic fibroblast growth factor (bFGF) and platelet

derived growth factor-b (PDGFb) were detected in serum by

immunoblotting using goat polyclonal anti-mouse antibodies to R-

spondin1 (1:200; R & D Systems, Minneapolis, MN), KGF (1:250),

bFGF (1:250) and PDGFb (1:250). Inflammatory cytokines were

measured in the serum using a multianalyte ELISArray kit (SA

Biosciences, Fredrick, MD), according to manufacturer’s protocol.

Cytokine and growth factors in crypt cellsTo compare the mRNA levels of different growth factors and

cytokines in intestine crypt cells from AIR and AIR+BMASCT

treated mice, real time PCR were performed using growth factor

(cat # PAMM-041) and cytokine (cat # PAMM-011) real time

array system from SA Biosciences.

Macrophage depletionTo deplete macrophages liposomal clodronate (Encapsula

NanoSciences, Nashville, TN, USA) (30 mg/kg of body weight)

was injected intravenously from day 4 pre-AIR to a week post-

AIR. Plain liposome was injected as control. Neither the

clodronate filled nor the empty liposomes are considered toxic to

the organs.

Inhibition of COX2NS-398 (Biomol, Plymouth Meeting, PA) was administered at a

dose of 1 mg/kg of body weight (36/week, ip) started at 1 week

prior to AIR. Animals treated with dmPGE2 (Sigma) received a

dose of 0.5 mg/kg of body weight (36/week, ip) started at 1 week

prior to AIR.

Kaplan-Meier Survival analysisMice survival/mortality in different treatment group was

analyzed by kaplan-Meier as a function of radiation dose using

Graphpad Prism-4.0 software for Mac.

Statistical analysis of digital imagesSampling regions were chosen at random for digital acquisition

for data quantitation. Digital image data was evaluated in a blinded

fashion as to any treatment. A two-sided student’s t-test was used to

determine significant differences between experimental cohorts

(P,0.05) with representative standard errors of the mean (SEM).

Supporting Information

Figure S1 Flowcytometric characterization of freshlyisolated bonemarrow cells for expression of MSCspecific (A) (CD105+ CD452) (B) (CD29+ CD452), (C)macrophage specific (CD11b+F480+) and (D) EPC spe-cific (CD133+ CD34+CD452) markers. It was noted that

bone marrow cell were primarily enriched with CD45+ hemato-

poetic cells (A–B).

(TIF)

Figure S2 Blood count was performed with the help ofANTECH DIAGNOSTICS (LAKE SUCCESS, NY) toevaluate the effect of abdominal irradiation (AIR) onhematopoesis. Absence of any significant changes in (A)

differential count and (B) number of RBC and among the

irradiated and transplanted group in comparison to untreated

control group suggested AIR could not affect the bone marrow.

(TIF)

Figure S3 Expression of different MSC surface markersCD105, CD29, CD44, SCA1 in BMASC population.Staining for IgG isotype fluorescence was used as a control.

Isotype control for CD105, CD29, CD44 and SCA-1 are rat

IgG2a, hamster IgG, rat IgG2bK and rat IgG2aK respectively.

(TIF)

Figure S4 Flowcytometric charaterization of CD11b2ve(A–B) and CD11b+ve (C–D) BMASC population forCD105 and CD29 (MSC marker) expression. It was noted

that CD11b2ve BMASC population was primarily enriched with

CD105 and CD29 positive cells.

(TIF)

Figure S5 BMASC transplantation significantly increas-es crypt depth compared to AIR control.

(PDF)

Figure S6 Kaplan-Meier survival analysis. Mice (n = 15)

receiving first dose of BMASC at 72 h post AIR follwed by second

dose failed to mitigate RIGS in contrast to BMASCT at 24 h

follwed by 72 hr second where 100% survival were noted.

(TIF)

Figure S7 Transplanted BMASC were primarily detect-ed in intestine and lung. BMASC from DPPIV positive wild

type mice were transplanted to DPPIV negative mice exposed to

AIR. (A&C) DPPIV immunohistochemistry followed by confocal

micrscopic analysis. DPPIV positive BMASC (stained green) were

found primarily in the lung (C) and intestine (A). Nucleus was

stained with DAPI and pseudo colored with red. (B&D)

Quantification of engrafted DPPIV+ve cells. Significantly higher

number of engrafted cells in lung (p,0.002) (B) and in intestine

(p,0.004) (D) was noted at 1day post AIR compared to 3.5 day

post AIR. Confocal microscopic images (636) were magnified

2.36and presented in inset. The number of DPPIV positive cells

were counted using volocity soft version 5 (Improvision). Based on

the intensities, number of cells were determined by scoring at least

10 fields from each animal (n = 3). Resolution of the images were

same for both experimental and control groups.

(TIF)

Figure S8 Immunoblot analysis of intestinal growthfactors in serum. An increase in the serum levels of R-

Stromal Cell Transplantation Mitigates RIGS

PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24072

spondin1, FGF2, KGF and PDGF-B was noted in AIR+BMAST

treated animals, compared to animals that received AIR+BM or

AIR alone.

(TIF)

Figure S9 A–B. Multi cytokine ELISA. A. Anti-inflammato-

ry cytokines, IL6 (p,0.004) and IL10 (p,0.002) levels were

significantly increased in the AIR+BMASCT-treated animals,

compared to AIR alone. Induction of anti-inflammatory cytokine

IL6 (p,0.007) and IL10 (p,0.005) was also observed in the

animals treated with AIR+CD11b+ve BMASCT. Transplantation

of freshly isolated bone-marrow cells could not increase the anti-

inflammatory cytokine level. B. AIR+BMASCT reduces the pro-

inflammatory cytokine levels (IL12A, IL17), compared to AIR

alone. Transplantation of freshly isolated bone-marrow cells could

not reduce the pro-inflammatory cytokine level compare to AIR

alone. C. AIR+BMASCT induces the GMCSF and GCSF levels

compared to AIR alone. Transplantation of freshly isolated bone-

marrow cells did not induce the GMCSF and GCSF level.

(TIF)

Figure S10 BMSCT maintains villus length after radia-tion injury. Low magnification images (106) of jejunal cross-

sections showed the reduction of villi length and thickness (H&E

staining) with the decrease in Brdu positive crypt cells in irradiated

cohort (18 Gy AIR) compared to 18 Gy+BMASC group.

(TIF)

Table S1 qPCR analysis of different growth factormRNA level in intestinal crypt cells. RT+BMASCT treated

group showed significant increase in mRNA level of growth factors

compared to RT cohort.

(DOC)

Table S2 qPCR analysis of inflammatory cytokine inintestinal crypt cells. RT+BMASCT treated group showed

significant increase in mRNA level of anti-inflammatory cytokine

level compared to RT cohort.

(DOC)

Table S3 Median survival time of animals exposed to18 Gy AIR and 10.4 Gy WBI followed by cell transplan-tation. Please note the clear difference of median survival time of

the animals exposed to 18 Gy AIR compared to 10.4 Gy WBI.

(DOC)

Text S1

(DOC)

Author Contributions

Conceived and designed the experiments: SS PB RK LL AA CG.

Performed the experiments: SS PB RK LL. Analyzed the data: SS PB RK

CG. Contributed reagents/materials/analysis tools: SS PB RK CG. Wrote

the paper: SS PB AA CG.

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